Cellular Localization of Clathridimine, an Antimicrobial 2-Aminoimidazole Alkaloid Produced by the Mediterranean Calcareous Sponge Clathrina Clathrus

J. Nat. Prod. XXXX, xxx, 000 A

Cellular Localization of Clathridimine, an Antimicrobial 2-Aminoimidazole Alkaloid Produced by the Mediterranean Calcareous Sponge Clathrina clathrus

Me´lanie Roue´,† Isabelle Domart-Coulon,‡ Alexander Ereskovsky,§,⊥ Chakib Djediat,| Thierry Perez,⊥ and Marie-Lise Bourguet-Kondracki*,† Laboratoire Molećules de Communication et Adaptation des Micro-organismes, FRE 3206 CNRS, Museúm National d’Histoire Naturelle, 57 Rue CuVier (C.P. 54), 75005 Paris, France, Laboratoire de Biologie des Organismes et des Ecosyste`mes Aquatiques, UMR 7208 MNHN-CNRS-IRD-UPMC, Museúm National d’Histoire Naturelle, 57 Rue CuVier (C.P. 26), 75005 Paris, France, Biological Faculty, Saint-Petersburg State UniVersity, UniVersitetskaya nab. 7/9, 199034 Saint-Petersburg, Russian Federation, Centre d’Oceánologie de Marseille, UMR 6540 DIMAR CNRS-UniVersite´delaMe´diterraneé, Station Marine d’Endoume, Rue de la Batterie des Lions, 13007 Marseille, France, and SerVice de Microscopie Electronique, Museúm National d’Histoire Naturelle, 57 Rue CuVier (C.P.39), 75005 Paris, France

ReceiVed March 12, 2010

Chemical investigation of the Mediterranean calcareous sponge Clathrina clathrus led to the isolation of large amounts of a new 2-aminoimidazole alkaloid, named clathridimine (1), along with the known clathridine (2) and its zinc complex (3). The structure of the new metabolite was assigned by detailed spectroscopic analysis. Clathridimine (1) displayed selective anti-Escherichia coli and anti-Candida albicans activities. Clathridine (2) showed only anti-Candida albicans activity, and its zinc complex (3) exhibited selective anti-Staphylococcus aureus activity. The isolation of analogues of 2-amino-imidazole derivatives from several Leucetta species from various sites in the Paciﬁc Ocean and the Red Sea raises the question of their biosynthetic origin. Microscopic studies revealed abundant extracellular bacteria located in the mesohyl of the sponge, with two predominant morphotypes including spiral bacteria and long, narrow bacilli. Chemical analysis with HPLC/UV/ELSD proﬁles of sponge cells separated from bacteria by differential centrifugation and trypsinization of the sponge cell surface revealed that clathridine (2) was localized in the sponge cells.

Marine sponges are a rich source of structurally unique natural cytotoxic,23-25 anti-inflammatory,30 and antitumor activities.31 Most compounds, with many of them displaying a wide variety of biological of them have been isolated from species belonging to the genus activities.1 They are also known to host a wide diversity of microor- Leucetta (Haeckel 1872), in the subclass Calcinea, order Clathrinida, ganisms that can amount up to 40-60% of the biomass of the sponge and family Leucettidae in specimens collected from various 2-4 and exceed seawater concentrations by 2-4 orders of magnitude. geographical sites (Pacific Ocean and Red Sea). These compounds Recently, these associations have raised questions about the origins have also been isolated from nudibranchs of the genus Notodoris, 5,6 of the metabolites isolated from sponges. However, despite this which feed on these sponges and sequester the alkaloid pigments growing interest in marine symbiotic association research, the complex in their dorsal mantle.16-18,31 To date there is no hypothesis interactions between sponges and associated microsymbionts remain regarding their biosynthesis, but their isolation from several Leucetta difficult to investigate. Thus the putative origin of natural compounds species from various sites raises the question of their biosynthetic isolated from sponges is often inferred from cellular localization - origin. Currently, there is little microbiological information available data.7 12 on sponges belonging to the Clathrinidae family. There is only one As part of our ongoing investigations on sponges within the ultrastructural report of a homogeneous population of one mor- framework of the French program ECIMAR (Ecologie Chimique: phological type of Gram-negative elongated bacterium inhabiting Indicateurs de Biodiversite´ et Valorisation), we studied the chem- the aquiferous canal system of Clathrina cerebrum (Haeckel, 1872) istry and microbiology of the Mediterranean calcareous sponge and of rare coiled bacteria associated with choanocytes of Clathrina Clathrina clathrus (Schmidt, 1864) (Calcispongia, Calcinea, Clath- 33 rinida, Clathrinidae). Unlike Demospongiae, Calcispongia have been clathrus. minimally studied in terms of both chemistry and microbiology. The 1:1 CH2Cl2/MeOH extract of C. clathrus revealed interesting Furthermore, their relatively low microbial density and diversity, broad-spectrum antimicrobial activities against both Gram-positive as reported for example for the Calcinea species Pericharax (Staphylococcus aureus) and Gram-negative (Escherichia coli) heteroraphis (Pole´jaeff, 1883)3,13 and for the Calcaronea species bacterial strains and against the yeast Candida albicans. The HPLC/ 2,13 Grantia compressa (Fabricius, 1780), might represent an UV/ELSD/MS profile of the 1:1 CH2Cl2/MeOH extract revealed advantage to better understand the origin of the secondary the presence of four metabolites belonging to the 2-aminoimidazole metabolites they produce and the putative role of their endosymbionts. alkaloid family: the new clathridimine (1), the known clathridine Calcareous sponges of the Calcinea subclass are known as sources (2), and two minor metabolites, namely, clathridine-zinc complex of 2-aminoimidazole alkaloids with more than 40 reported compounds (3) and preclathridine (4). including clathridines,14-17 naamidines and isonnamidines,17-25 and 18,19,21,22,26-29 The current report describes the isolation and purification of naamines and isonaamines. Some of these alkaloids - have promising biological activities such as antifungal,14,21 compounds 1 3, the structure elucidation of clathridimine (1), and the antimicrobial activities of 1-3. Furthermore, due to the detection of an abundant bacterial community within the sponge, morphologi- * To whom correspondence should be addressed. Tel: +33 140 795 606. Fax: +33 140 793 135. E-mail: [email protected]. cally characterized with scanning (SEM) and transmission (TEM) † Laboratoire Molećules de Communication et Adaptation des Micro- electron microscopy, we investigated the cellular localization of organismes. these secondary metabolites. Chemical profiles of populations ‡ Laboratoire Biologie des Organismes et des Ecosyste`mes Aquatiques. enriched either in bacteria or in cell types of C. clathrus, separated § Saint-Petersbourg State University. ⊥ Centre d’Oceánologie de Marseille. by differential centrifugation and trypsinization of the sponge cell | Service de Microscopie Electronique. surface, are presented.

10.1021/np100175x  XXXX American Chemical Society and American Society of Pharmacognosy B Journal of Natural Products, XXXX, Vol. xxx, No. xx Roue´ et al.

Figure 1. HPLC/ELSD chromatogram of the CH2Cl2/MeOH extract of Clathrina clathrus.

Table 1. NMR Spectroscopic Data (CDCl3) for Clathridimine (1) a no. δC, mult. δH (J in Hz) HMBC 2 147.6, C Results and Discussion 4 133.9, C 5 115.9, CH 6.39, s 2, 4 The HPLC/ELSD chromatogram of the 1:1 CH2Cl2/MeOH 7 n.o.b extract (Figure 1), after desalinization on a C18 SPE column, 9 158.5, C revealed the presence of two major compounds, 1 and 2. Successive 11 163.2, C chromatographies of the extract on reversed-phase columns led to 12 32.2, CH3 3.67, s 2, 5 the isolation of three 2-aminoimidazole alkaloids, 1-3, belonging 13 25.6, CH3 3.17, s 9, 11 ′ ′ ′ to the clathridine family, as first suggested by the UV profiles. The 14 32.8, CH2 3.88, s 4, 5, 1 ,2,6 1′ 131.2, C structures of these natural products were determined by mass 2′ 109.1, CH 6.72, s 14, 3′,4′,6′ analysis and 1D and 2D NMR spectroscopic studies. Preclathridine 3′ 147.9, C (4), previously isolated from the nudibranch Notodoris gardineri,16 4′ 146.6, C was also observed in the sponge extract, as deduced from LC/MS 5′ 105.4, CH 6.76, d (7.8) 1′,3′,4′ experiments, but the molecule was present in an amount too small 6′ 121.9, CH 6.70, d (7.8) 14, 2′,4′ ′ ′ ′ to be isolated. 7 101.0, CH2 5.93, s 3 ,4 Compound 2 was obtained as yellow crystals. Its molecular a HMBC correlations, optimized for 7 Hz, are from proton(s) stated to the indicated carbon. b n.o.: signal not unambiguously observed. formula C16H15N5O4 was obtained from its ESIMS, which showed the pseudomolecular ion [M + H]+ at m/z 342.1189, indicating the presence of 12 unsaturations in the molecule. Detailed examina- 163.2), were also reminiscent of clathridine (2) (Table 1). Com- tion of the spectroscopic data readily established its identity as parison of all these data indicated that in 1 one carbonyl group clathridine, an antifungal compound previously isolated from C. was replaced by one imino group. Its localization, out of the two 14 clathrus. possibilities (C-9 or C-11), was solved by the resonance at δC 158.5 Compound 1 was obtained as a yellow-orange solid. ESIMS corresponding to the carbon bearing the imino group in position showed the pseudomolecular ion [M + H]+ at m/z 341.1354, which C-9, as previously reported for (2E,9E)-pyronaamidine 9-(N- differed from that of clathridine (2) by only one dalton, and led to methylimine) (5), in which the ambiguity in the structure was 25 the molecular formula C16H16N6O3, implying the presence of 12 resolved through X-ray single-crystal diffraction analysis. There- unsaturations in the molecule. Comparison of its 1H NMR and 13C fore, the structure of compound 1, named clathridimine, was NMR spectroscopic data with those of clathridine 2 confirmed established as being a new 2-aminoimidazole alkaloid with an imino strong similarities between the two compounds. Compound 1 group at the C-9 position. possessed a similar piperonyl unit, as indicated in its 1H NMR Compound 3 was obtained as yellow crystals. Its ESI mass + + spectrum (CDCl3) with the presence of two methylene singlets at spectrum showed a molecular ion cluster [M H] at m/z 745, δ 3.88 and 5.93 and three aromatic protons at δ 6.72 (s) and at δ 747, and 749 (relative intensities 100, 60 and 33), clearly indicative 6.70 (d, J ) 7.8 Hz) and 6.76 (d, J ) 7.8 Hz), which was confirmed of the presence of one zinc atom in the molecule. Examination of with 2D NMR data. An additional singlet methine proton at δ 6.39 spectroscopic data identified compound 3 as the Zn complex of 14,34 (δC 115.9) and singlet N-methyl protons at δ 3.67 (δC 32.2) and clathridine (2), previously described in C. clathrus. 13 3.17 (δC 25.6), supported by further resonances in the C NMR The 1:1 CH2Cl2/MeOH extract showed a broad spectrum of spectrum of quaternary carbons at δC 147.6 and 133.9 corresponding activities against the bacterial strains Staphylococcus aureus and 2 to two fully substituted sp carbon atoms and one carbonyl (δC Escherichia coli and the yeast Candida albicans (Table 2). The Cellular Localization of Clathridimine Journal of Natural Products, XXXX, Vol. xxx, No. xx C

a Table 2. Antimicrobial Activities of the 1:1 CH2Cl2/MeOH be transformed into clathridine (2) during the cell fractionation Extract of the Sponge C. clathrus and of Compounds 1-3 procedure. This suggestion was supported by hydrolysis of clath- S. aureus E. coli C. albicans ridimine (1) into clathridine (2) overnight by addition of water in ATCC 6538 ATCC 8739 ATCC 66029 excess. Therefore, due to its instability in water, the origin of clathridimine (1) could not be determined. Clathridine (2) was CH2Cl2/MeOH extract 11 ( 215( 320( 1 of C. clathrus localized in the sponge cell fractions (C1 and C1tryp) and not in the clathridimine (1)0 15( 224( 4 bacterial fractions (C3 and C3tryp) (Figure 5). Traces of clathridine clathridine (2)0018( 1 (2) were also detected in fraction C2, due to the presence of some clathridine Zn 12 ( 10 0 remaining sponge cells, and in both fraction C3 and supernatant S, complex (3) which might be explained by lysis of sponge cells during mechanical kanamycin 37 ( 438( 4 itraconazole 21 ( 1 dissociation and centrifugation (Figure 5). The enrichment in sponge a cells was linked to the increase in clathridine content, as shown by Inhibition diameter measured by the agar diffusion assay method for the comparative size of peaks on HPLC chromatograms. The 1 mg per well (6 mm of diameter) for the extract and 100 µg per well for pure compounds and controls. Samples were tested in triplicate. clathridine-zinc complex (3) was localized, through LC/MS experiments, only in the supernatant, suggesting its possible excretion. three 2-aminoimidazole alkaloids 1-3 were tested against these In conclusion, a new 2-aminoimidazole alkaloid, named clath- three microorganisms at 100 µg/well. They revealed different and ridimine (1), along with the known clathridine (2) and its zinc selective activities. Clathridine (2) displayed specific antifungal complex (3), has been isolated from the Mediterranean calcareous activity against C. albicans, and its Zn complex (3) exhibited sponge C. clathrus with specific activities against various micro- specific anti-S. aureus activity. The new clathridimine (1) inhibited organisms. Although clathridimine (1), unstable in water, could not growth of the bacterial pathogen E. coli and the fungal pathogen be localized, clathridine (2) proved to be associated with the sponge C. albicans. Although qualitative, these results might suggest a role cells through differential centrifugation. However, this experiment for these compounds as antimicrobial chemical defense of C. provides only circumstantial evidence because the hypothesis of clathrus. transport of clathridine from one cell type to another cannot be 37 Light microscopic, SEM, and TEM studies revealed that C. excluded. Future studies will investigate the ecological role of clathrus contained numerous extracellular bacteria dispersed in the these compounds against environmental fungi and bacteria. mesohyl of the sponge body wall (Figure 2B). Bacterial abundance was estimated to be (2.2 ( 0.7) × 108 bacteria/g sponge wet weight Experimental Section 8 (n ) 3 specimens) as compared to a total of (1.5 ( 0.7) × 10 General Experimental Procedures. UV spectra were recorded on sponge cells/g sponge wet weight (n ) 3 specimens) counted in a UVIKON 930 spectrometer. IR spectra were recorded on a FT-IR Dapi-DNA-stained dissociated sponge suspensions. Choanocytes Shimadzu 8400 S spectrometer. 1H NMR 1D and 2D spectra (COSY, were the major (70%) sponge cell types in C. clathrus. Intracellular HSQC, HMBC, NOESY) were obtained on a Bruker AVANCE 400. bacteria were not detected ultrastructurally in sponge cell types. HSQC and HMBC experiments were acquired at 400.13 MHz using a 1 13 13 Two preponderant morphological types of extracellular bacteria H- C dual probehead. The delay preceding the C pulse for the were localized in the mesohyl: curved, spiral bacteria (Figures 2C creation of multiple quanta coherences through several bounds in the and D) and long, narrow rods (Figures 2E and F). These bacteria HMBC was set to 70 ms. HMBC spectra were optimized for 7 Hz coupling. Mass spectra were recorded on an API Q-STAR PULSAR I were sometimes observed in close proximity to the sponge cell from Applied Biosystem. LC/MS analyses were carried out with a surface (Figure 2D). Jupiter column (Phenomenex, 5 µm, C18, 300A, 150 × 1.0 mm). The In order to determine the cellular localization of these secondary extract was desalted on a SPE Strata C18-E column (55 µm, 70A). metabolites, sponge cells were separated from bacteria via dif- Semipreparative (Gemini C6-phenyl, 10 × 250 mm) and analytical ferential sedimentation of dissociated sponge suspensions (Figure (Gemini C6-phenyl, 3 × 250 mm) reversed-phase HPLC were 3). Cell pellets from centrifugation at three different speeds were performed with an Alliance apparatus (model 2695, Waters) equipped with a photodiode array detector (model 2998, Waters), an evaporative lyophilized and extracted with CH2Cl2/MeOH, and each extract was analyzed by HPLC/UV/ELSD chromatography and by LC/MS light-scattering detector (model Sedex 80, Sedere), and the software Empower. experiments. Microscopic observations showed that this successive Animal Material. Specimens of Clathrina clathrus were collected pelleting method was appropriate to obtain fractions enriched either by scuba diving in the northwestern Mediterranean Sea (Marseille, in sponge cells (heavy fraction, C1) or in bacteria (bacterial fraction, France) from July 2007 to November 2009 between 10 and 15 m depth. C3) (Figure 4). The heavy fraction, C1, contained mostly sponge A voucher specimen is available from the Museum National d’Histoire cells (choanocytes, a few larger spherulous cells, and undetermined Naturelle in Paris as Porifera collection number C2010-1. sponge cell types) (Figure 4 panel C1) with remaining bacteria Extraction and Isolation of Compounds 1, 2, and 3. Freshly closely associated with the cell surface. The light fraction, C2, collected animal (wet weight: 40 g) was lyophilized (4.2 g), stored × contained mostly bacteria with some remaining sponge cells (Figure frozen, and further extracted with 1:1 CH2Cl2/MeOH (3 20 mL, 4 panel C2). The bacterial fraction, C3, contained exclusively sonication for 15 min) at room temperature. The 1:1 CH2Cl2/MeOH extract was concentrated under reduced pressure to yield a dark brown, bacteria and potential cell debris (Figure 4 panel C3). Heavy fraction viscous oil (208.1 mg), which was chromatographed on a C18 SPE C1 was enriched in sponge cells by a factor of 9 compared to light column in a vacuum chamber (H O, H O/MeOH, 1:3, MeOH, CH Cl , fraction C2 and by a factor of 30 compared to bacterial fraction 2 2 2 2 100 mL of each). The fraction that eluted with H2O/MeOH, 1:3 (47.9 C3. Trypsinization of the sponge cell surface allowed improved mg), was subjected to semipreparative reversed-phase HPLC (Gemini separation of sponge cells from extracellular bacteria that may be C6-phenyl 10 × 250 mm) with increasing amounts of CH3CN/0.1% closely associated with the sponge cell surface, as proven by the formic acid in H2O/0.1% formic acid as eluent (flow rate: 5 mL/min, low amount of bacteria in the pellet C1tryp (containing 10 times wavelength: 254 and 280 nm) and afforded clathridimine (1) (4.6 mg, more sponge cells than bacteria) compared to the high enrichment 0.11% of sponge dry weight), clathridine (2) (2.8 mg, 0.07% of sponge dry weight), and the zinc complex of clathridine (3) (1.4 mg, 0.03% of of the pellet C3tryp in bacteria with almost no sponge cells. sponge dry weight). The results were unequivocal and showed that clathridimine (1) Clathridimine (1): yellow-orange solid; UV (EtOH) λmax (log ε) was found only in the CH2Cl2/MeOH extract of the whole sponge 284 (3.73), 375 (3.84) nm; IR (dry film) νmax 3240, 1775, 1740, 1694, before dissociation and was totally absent from the other extracts 1620, 1559, 1489, 1446, 1397, 1303, 1246, 1114, 1038, 926 cm-1; 1H (Figure 5). These results suggested that clathridimine (1)isa and 13C NMR data, see Table 1; ESIMS m/z 341.1354 [M + H]+ (calcd genuine compound produced by the sponge C. clathrus and may for C16H17N6O3, 341.1356). D Journal of Natural Products, XXXX, Vol. xxx, No. xx Roue´ et al.

Figure 2. Light micrographs (A, B) of Gram-stained histological sections, showing ascon organization of the aquiferous system of Clathrina clathrus (A) with magniﬁcation of the thin mesohyl portion of the tubes containing numerous bacteria (B). Transmission electron micrographs (C and E) and scanning electron micrographs (D and F) of the two preponderant bacterial morphotypes: curved, spiral bacteria (C and D) and long, narrow rods (E and F). c: choanoderm, m: mesohyl, p: pinacoderm.

Figure 3. Localization of clathridine (2) in the sponge cell enriched fractions (C1, C1tryp), obtained by differential centrifugation and sponge cell surface trypsinization. Clathridine (2): yellow crystals; spectroscopic data matched those at pH 4 (5% acetic acid 75% saturated picric acid, and 20% formol), previously published.13 then washed extensively in 70% EtOH and stored at 4 °C. Fixed tissue Zn Complex of Clathridine (3): yellow crystals; spectroscopic data was dehydrated in an increasing series of EtOH substituted by xylene matched those previously published.13,33 and embedded in paraffin. Histological sections (5-6 µm, Microtome Hydrolysis of Clathridimine (1) into Clathridine (2). Compound Leitz) were deparaffinized in xylene and rehydrated and were Gram 1 (200 µg) was treated with 20 mL of water overnight at room stained (first stained with crystal violet for 3 min, then covered with temperature and then analyzed by ESIMS. Lugol’s solution for 3 min, differentiated with 95% EtOH, and Antimicrobial Assays. Assays were performed by the agar diffusion counterstained with Ziehl fuchsin for 3 min). Tissue sections were assay method. Extract (1 mg) or pure compound (100 µg) was observed by light microscopy with a DMLB microscope fitted with a solubilized in DMSO and deposited ina6mmwell cut out from agar Leica DC300F camera, and images were acquired with the IM500 plates seeded with reference strains Staphylococcus aureus (ATCC software. For transmission electron microscopy (TEM) and scanning 6538), Escherichia coli (ATCC 8739), and Candida albicans (ATCC electron microscopy (SEM), pieces of sponges were fixed immediately 66029). Antimicrobial activity was determined by measuring the after collection in 2.5% glutaraldehyde in a mixture of 0.4 M cacodylate diameter of the inhibition zones after 24 h of incubation at 37 °C for buffer and seawater (1:4:5; 1120 mOsm, pH 7.4), postfixed in 2% OsO4 S. aureus and E. coli or 30 °C for C. albicans. Kanamycin was used as in seawater, and dehydrated through a graded EtOH series. Alternately, the standard antibiotic positive control for the S. aureus and E. coli sponge fragments were fixed in 2.5% glutaraldehyde in So¨rensen bacterial strains and itraconazole for the C. albicans yeast. phosphate 0.1 M/sucrose 0.6 M buffer (pH 7.6) containing 10 mM Microscopic Studies. For light microscopy, fragments of sponges CaCl2 and 0.055% w/v ruthenium red, postfixed in 1% OsO4 in were fixed and decalcified overnight after collection in Bouin fixative So¨rensen buffer, and dehydrated in EtOH. For SEM, specimens Cellular Localization of Clathridimine Journal of Natural Products, XXXX, Vol. xxx, No. xx E

Figure 4. Cell type composition of the fractions obtained by differential centrifugation and stained with methylene blue-azur II: C1, heavy fraction (enriched in sponge cells); C2, light fraction (enriched in bacteria, with a few sponge cells); C3, bacterial fraction. Arrows point to large spherulous sponge cell types.

Figure 5. HPLC/ELSD chromatograms of the CH2Cl2/MeOH extracts (injection of 200 µg) of whole sponge and of fractions C1 (heavy sponge cells), C2 (mixed bacteria and small-sized sponge cells), C3 (bacteria), S (supernatant), C1tryp (heavy sponge cells), and C3tryp (bacteria detached from sponge cell surface). dehydrated in EtOH were fractured in liquid nitrogen, critical-point- To check the efficiency of the cell-bacteria separation protocol, a dried, sputter-coated with gold-palladium, and observed under a part of the dissociated sponge suspension and of each fraction was fixed Hitachi S570 SEM. For TEM the pieces were embedded in Araldite or with paraformaldehyde at 3% final concentration or with glutaraldehyde Spu¨rr resin. Sections were cut with a Diatome 45° diamond (Ultracut 2.5% final concentration and stored at 4 °C until further processing. microtome). Semithin sections were stained with toluidine blue 1%/ After elimination of the fixative by centrifugation, the cells and/or borax 1% in 70% ethanol. Ultrathin sections were counterstained with bacteria were resuspended in PBS/EtOH/Tween 20 50/50/0.1% (pH uranyl acetate 2% in 50% and were observed under a Zeiss-1000 TEM 7.4) to help dissociate small aggregates. The resulting suspensions were and LEO 910 at 75 kV with a Hitachi H7100 transmission electron smeared on a glass slide, and the DNA was stained with the microscope equipped with a digital CCD Hamamatsu camera. fluorochrome DAPI 0.1% in H O, or the cells were visualized either Sponge Dissociation and Cell Fractionation. Freshly collected 2 with methylene blue/azur II or Groat hematoxylin histological stains sponges were rinsed with artificial seawater, and their epibionts were according to Martoja et al.36 Alternately, suspensions were stained with carefully discarded. Sponge fragments were mechanically dissociated by pressing through a 40 µM pore-size autoclaved plankton net in cold DAPI 0.1% in PBS/EtOH/Tween 20 50/50/0.1% (pH 7.4), and sponge (4 °C) calcium and magnesium-free artificial seawater (CMF-ASW), cells and bacteria were counted on a Petroff-Hausser slide according according to Wilson.35 The suspension was then filtered through a 40 to the manufacturer’s directions, with a DMLB epifluorescence µm mesh sterile nylon sieve to remove large spicule debris and cell microscope under blue light (Leica Microsyste`mes SAS). The abun- aggregates. dance of cells versus bacteria was visualized by the difference in size In order to separate populations enriched either in cell types of C. of marked nuclei versus marked bacterial DNA. clathrus or in bacteria, the cell suspension obtained by mechanical The remainder of the different fractions were frozen without fixation, dissociation was centrifuged first at 200g for 10 min (pellet C1 enriched lyophilized, extracted with 1:1 CH2Cl2/MeOH, and desalted on SPE in sponge cells), then its supernatant at 500g for 10 min (pellet C2 C18 for HPLC chromatography (injection of 200 µg of each) and for with bacteria and some remaining sponge cells), and finally the second LC/MS experiments in order to look for the presence of clathridine 2 supernatant at 10000g for 10 min (pellet C3 enriched in bacteria), and its analogues. according to a protocol modified from Richelle-Maurer et al.11 In order to separate extracellular bacteria potentially associated with the cell Acknowledgment. This work is part of M. Roué’s thesis, within surface in the C1 heavy fraction, a trypsinization experiment was the French program ECIMAR, supported by the Agence Nationale pour conducted. The pellet C1 enriched in sponge cells was resuspended in CMF-ASW and trypsinized (30 mg of trypsin/g of sponge wet weight) la Recherche (ANR). We thank A. Blond and A. Deville (MNHN, Paris) for 15 min at room temperature (20 °C). The suspension was centrifuged for NMR spectra, A. Marie and L. Dubost (MNHN, Paris) for MS at 200g for 10 min to separate the cells (C1tryp enriched in sponge cells) measurements, M. Martin for preparation of histological sections, and from the supernatant, which was then centrifuged at 10000g for 10 M. Vandervennet for her help in antimicrobial bioassays. min (C3tryp enriched in bacteria detached from sponge cell surface). The cell-bacteria separation and trypsinization experiments were Supporting Information Available: NMR and ESIMS spectra of repeated three times independently, with two replicates per experiment. 1 are available free of charge via the Internet at http://pubs.acs.org. F Journal of Natural Products, XXXX, Vol. xxx, No. xx Roue´ et al.

References and Notes (20) Mancini, I.; Guella, G.; Debitus, C.; Pietra, F. HelV. Chim. Acta 1995, 78, 1178–1184. (1) Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.; Prinsep, (21) Fu, X.; Schmitz, F. J.; Tanner, R. S.; Kelly-Borges, M. J. Nat Prod. M. R. Nat. Prod. Rep. 2010, 27, 165–237. 1998, 61, 384–386. (2) Vacelet, J.; Donadey, C. J. Exp. Mar. Ecol. 1977, 30, 301–314. (22) Gross, H.; Kehraus, S.; Ko¨nig, G. M.; Wo¨rheide, G.; Wright, A. D. J. (3) Wilkinson, C. R. Mar. Biol. 1978, 49, 161–167. Nat. Prod. 2002, 65, 1190–1193. (4) Hentschel, U.; Usher, K. M.; Taylor, M. W. FEMS Microbiol. Ecol. (23) Tsukamoto, S.; Kawabata, T.; Kato, H.; Ohta, T.; Rotinsulu, H.; 2006, 55, 167–177. Mangindaan, R. E. P.; van Soest, R. W. M.; Ukai, K.; Kobayashi, H.; (5) Haygood, M. G.; Schmidt, E. W.; Davidson, S. K.; Faulkner, D. J. J. Namikoshi, M. J. Nat. Prod. 2007, 70, 1658–1660. Mol. Microbiol. Biotechnol. 1999, 1, 33–43. (24) Akee, R. K.; Caroll, T. R.; Yoshida, W. Y.; Scheuer, P. J. J. Org. (6) Piel, J. Curr. Med. Chem. 2006, 13, 39–50. Chem. 1990, 55, 1944–1946. (7) Bewley, C. A.; Holland, N. D.; Faulkner, D. J. Experientia 1996, 52, (25) Plubrukarn, A.; Smith, D. W.; Cramer, R. E.; Davidson, B. S. J. Nat. 716–722. Prod. 1997, 60, 712–715. (8) Uriz, M. J.; Turon, X.; Galera, J.; Tur, J. M. Cell. Tissue Res. 1996, (26) Fu, X.; Barnes, J. R.; Do, T.; Schmitz, F. J. J. Nat. Prod. 1997, 60, 285, 519–527. 497–498. (9) Flowers, A. E.; Garson, M. J.; Webb, R. I.; Dumdei, E. J.; Charan, (27) Dunbar, D. C.; Rimoldi, J. M.; Clarck, A. M.; Kelly, M.; Hamann, R. D. Cell. Tissue Res. 1998, 292, 597–607. M. T. Tetrahedron. 2000, 56, 8795–8798. (10) Richelle-Maurer, E.; Braekman, J. C.; De Kluijver, M. J.; Gomez, R.; (28) Crews, P.; Clark, D. P.; Tenney, K. J. Nat. Prod. 2003, 66, 177–182. van de Vyver, G.; van Soest, R. W. M.; Devijver, C. Cell. Tissue (29) Hassan, W.; Edrada, R. A.; Ebel, R.; Wray, V.; Berg, A.; van Soest, Res. 2001, 306, 157–165. R.; Wiryowidagdo, S.; Proksh, P. J. Nat. Prod. 2004, 67, 817–822. (11) Salomon, C.; Deerinck, T.; Ellisman, M.; Faulkner, D. Mar. Biol. 2001, (30) Chan, G. W.; Mong, S.; Hemling, M. E.; Freyer, A. J.; Offen, P. H.; 139, 313–319. Debrosse, C. W.; Sarau, H. M.; Westley, J. W. J. Nat. Prod. 1993, (12) Flowers, A. E.; Garson, M. J.; Webb, R. I.; Dumdei, E. J.; Charan, 56, 116–121. R. D. Cell. Tissue Res. 1998, 292, 597–607. (31) Copp, B. R.; Fairchild, C. R.; Cornell, L.; Casazza, A. M.; Robinson, (13) Hentschel, U.; Fieseler, L.; Wehrl, M.; Gernert, C.; Steinert, M.; S.; Ireland, C. M. J. Med. Chem. 1998, 41, 3909–3911. Hacker, J.; Horn, M. In Marine Molecular Biotechnology;Mu¨ller, (32) Alvi, K. A.; Crews, P. J. Nat. Prod. 1991, 54, 1509–1515. W. E. G., Ed.; Springer-Verlag: Berlin, 2003; pp 59-88. (33) Burlando, B.; Sabatini, M. A.; Gaino, E. J. Exp. Mar. Biol. Ecol. 1988, (14) Ciminiello, P.; Fattorusso, E.; Magno, S.; Mangoni, A. Tetrahedron 116, 35–42. 1989, 45, 3873–3878. (34) Ciminiello, P.; Fattorusso, E.; Mangoni, A. Tetrahedron 1990, 46, (15) He, A. Y.; Faulkner, D. J. J. Org. Chem. 1992, 57, 2176–2178. 4387–4392. (16) Alvi, K. A.; Peters, B. M.; Hunter, L. M.; Crews, P. Tetrahedron 1993, (35) Wilson, H. V. J. Exp. Zool. 1907, 5, 245–258. 49, 329–336. (36) Martoja, R.; Martoja, M. Initiations aux Techniques de l’Histologie (17) Carroll, A. R.; Bowden, B. F.; Coll, J. C. Aust. J. Chem. 1993, 46, Animale; Masson et Cie.: Paris, 1967; p 345. 1229–1234. (37) Simmons, T. L.; Coates, R. C.; Clark, B. R.; Engene, N.; Gonzalez, (18) Carmely, S.; Kashman, Y. Tetrahedron Lett. 1987, 28, 3003- D.; Esquenazi, E.; Dorrestein, P. C.; Gerwick, W. H. Proc. Natl. Acad. 3006. Sci. U. S. A. 2008, 105, 4587–4594. (19) Carmely, S.; Ilan, M.; Kashman, Y. Tetrahedron. 1989, 45, 2193– 2200. NP100175X